title: Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging.
abstract: Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Zeff) and nonelectrostatic (i.e., specific) component of binding, Kd(1M). Gag binds to Psi RNA with a dramatically reduced Kd(1M) and lower Zeff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Zeff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Zeff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Webb
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Jones
authors.forename: Leslie J
authors.initials: LJ
authors.lastname: Parent
authors.forename: Ioulia
authors.initials: I
authors.lastname: Rouzina
authors.forename: Karin
authors.initials: K
authors.lastname: Musier-Forsyth
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly / Q000235:genetics / Q000502:physiology; D016335:Zinc Fingers / Q000235:genetics; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058727:RNA, Small Untranslated
chemical_list: D012367:RNA, Viral; D054301:gag Gene Products, Human Immunodeficiency Virus; D058727:RNA, Small Untranslated
keywords: Gag; HIV; Psi; UTR; packaging signal